Methods of virus production

ABSTRACT

A simple yet effective method of increasing production of a thermo-stable virus, such as adenovirus and picornavirus, is presented. The method entails a temperature shift strategy whereby the culture of host cells are shifted to a suboptimal temperature for a period of time prior to virus infection or cells are grown at a suboptimal level for the entire cell expansion process including one or more than one passages of cell growth from cryopreserved cells, followed by a shift back to a more optimal temperature at or near the time of virus infection of the respective host cells. Adaptation of such a temperature shift strategy present a simple yet effective method to substantially increase recoverable virus within a respective host cell/virus production scheme without the need to further manipulate other culture and/or media conditions within an established host cell/virus production scheme.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit, under 35 U.S.C. §119(e), to U.S.provisional application 60/368,654 filed Mar. 29, 2002.

STATEMENT REGARDING FEDERALLY-SPONSORED R&D

Not Applicable

REFERENCE TO MICROFICHE APPENDIX

Not Applicable

FIELD OF THE INVENTION

The present invention relates to a method of maximizing production of athermo-stable virus based on a cell culture temperature shift strategywhich results in a substantial enhancement of thermal stable virusproduction. The manipulation of cell culture conditions calls forgrowing cells at a sub-optimal host cell growth temperature for a periodof time prior to virus infection, by shifting the cell growthtemperature down to a sub-optimal level for a period of time or bygrowing cells at a sub-optimal temperature for the entire multiplepassages of cell expansion process from the inoculation of acryopreserved ampule of cells, followed by shifting the temperature upto a higher level prior to, simultaneous to, or after virus infection ofthe host cells. This methodology results in a substantial increase inrecoverable virus as compared with known temperature schemes for virusproduction within a host cell culture.

BACKGROUND OF THE INVENTION

Advances in the areas of the use of recombinant viral vectors for genetherapy and DNA vaccination applications have created a need for largescale manufacture and purification of clinical-grade virus. One suchfamily of viruses are the adenoviruses. The adenoviruses are groupedwithin the family Adenoviridae, which are split into the genusAviadenovirus (birds) and Mastadenovirus (human, simian, bovine, equine,porcine, ovine, canine and opossum). A review of the family Adenoviridaecan be found in Fundamental Biology, 3^(rd) Ed., Fields, B. N., Knipe,D. M., and Howley, P. M., Ed., at Chapter 30, pp. 979-1016 (1996), whichis hereby incorporated by reference. Of specific interest in genevaccination and/or gene therapy applications is the use of a replicationincompetent adenovims, crippled by E1 or further deletions, including“gutless” adenovirus vectors. The adenovirus genome is generallyassociated with benign pathologies in humans, and the genomicorganization of the virus has been well studied since its discovery inthe early 1950s. In addition, the genome is amenable to manipulation,depending on the strategy utilized to construct the respective vector. Areplication-incompetent virus (such as an E1/E3 deleted Ad5gag vectorexpressing a HIV gag transgene, as exemplified herein) requires a cellline which complements the deletions. Any such cell line may be used togenerate recombinant virus vectors, with preferred, but not limiting,cell lines including 293 cells and PER.C6™ cells. To this end, numerous1^(st) generation recombinant adenovirus vectors have been described inthe literature (e.g., see Bett, et al., 1994, Proc. Natl. Acad. Sci.91:8802-8806; WO 01/02607 and WO 02/22080). “Gutless” adenoviral vectorsare a 2^(nd) generation adenoviral vector generally devoid of viralprotein-coding sequences, frequently with viral proteins supplemented intrans by a helper virus (often an E1-deleted adenovirus) grown with thehelper-dependent (HD) adenovector in a packaging cell line (e.g.,PER.C6™). Absent viral proteins, these viral vectors can, in thealternative, be supplemented in trans by a cell line and/or “helpervirus” capable of expressing the structural and functional adenoviralproteins necessary for successful replication, packaging and rescue. Inview of the increased popularity of these viral vectors and the ultimateneed to prepare commercial scale quantities of either a viral vectorbased vaccine or gene therapy vehicle, it has become essential todevelop more efficient qualitative and quantitative methodology forproduction of commercial grade recombinant adenovirus vectors.

It has been shown that temperature is an important process parameter forboth cell growth and virus production. The physiological temperature of37° C. has been shown to be optimal for growth of a majority ofmammalian cell lines. Temperatures below 37° C. historically are shownto reduce cell growth rate, overall cell metabolism, and specificproduct formation in mammalian cells (see, e.g., Moore, et al., 1997,Cytotechnology 23:47-54; Chuppa, et al., 1997, Biotechnol Bioeng.55:328-338). The optimal temperature for virus production depends on thevirus strain and the host cell line, but has most often been found to bebelow 37° C., including 34° C. for herpes simplex virus (HSV) productionin FL cell culture (Hoggan and Roizman, 1959, Virology 8:508-524), 32 to34° C. for myxoma virus (Ross and Sanders, 1979, J. Gen. Virol.43:213-216), and 35° C. for foot-and-mouth disease virus in suspensionBHK 21 cell cultures (Capstick et al., 1967, J. Hyg. Camb. 65:273-280),and 32° C. for cold adapted influenza viruses in MDCK cells.Temperatures above 37° C. are generally not suitable or evennon-permissive for virus replication (Schweitzer-Thumann et al., 1994,Res. Virol. 145:163-170). For highly heat-labile viruses such asretrovirus, virus productivity can be significantly increased byshifting the culture temperature down from 37° C. (during cell growth)to 32° C. for virus production, primarily due to increased virusstability at a lower temperature (McTaggart and Al-Rubeai, 2000,Biotechnol. Prog. 16:859-865).

Despite these reports, there remains a need for the development of alarge scale process for virus production from cell culture which addressboth quantitative and qualitative issues that are imposed upon acommercialized viral-based vaccine and/or gene therapy product. Thepresent invention addresses and meets these needs by disclosing anoptimized cell culture and virus production process which definesoptimal temperature ranges, resulting in an improved virus productivityas well as elimination of intra-batch productivity variations.

SUMMARY OF TEE INVENTION

The present invention relates to a method of maximizing production of athermo-stable virus based on a cell culture temperature shift strategywhich results in a substantial enhancement of thermal stable virusproduction. The manipulation of temperature within the cellculture/virus production process disclosed herein relies upon atemperature shift strategy whereby (1) the culture of host cells areshifted to a sub-optimal temperature for a period of time prior to virusinfection or, (2) the host cell culture is inoculated and grown at arespective sub-optimal temperature, followed by a shift back to a moreoptimal growth temperature at or near the time of virus infection of therespective host cells. Adaptation of such a temperature shift strategypresents a simple yet effective method to substantially increaserecoverable virus within a respective host cell/virus production schemewithout the need to further manipulate other culture and/or mediumconditions within an established host cell/virus production scheme.

While any virus which is amenable to production in a temperaturecontrolled cell culture environment is envisioned to fall within thescope of this disclosure, the present invention is especially applicableto thermo-stable viruses, such as members of the Adenoviridae family(including all known adenovirus serotypes, and recombinant virusgenerated from such an adenovirus serotype, including any first orsecond generation recombinant adenovirus vector known in the art) andmembers of the Picornavirus family (e.g., poliovirus, rhinovirus,hepatitis A virus, Foot and Mouth Disease Virus). A preferred virus isany serotype of adenovirus, and especially preferred is any recombinant1^(st) or 2^(nd) generation recombinant adenovirus vector containing atleast one heterologous transgene (see, e.g., e.g., see Bett, et al.,1994, Proc. Natl. Acad. Sci. 91:8802-8806; WO 01/02607 and WO 02/22080,the three publications hereby incorporated by reference).

As noted above, the present invention relies in part on a temperatureshift strategy for cell growth which comprises reducing the culturetemperature to a sub-optimal level for a period of time prior to virusinfection or growing cells at a sub-optimal level for the entire cellexpansion process including one or more than one passages of cell growthfrom cryopreserved cells, followed by a shift up to or near thephysiological temperature for production of that particular virus priorto, simultaneous to, or after virus infection of the host cells. Variousparameters which may be further manipulated in relation to thetemperature shift methodology disclosed herein and which fall within thescope of the present invention include but are not necessarily limitedto (1) altering the range of the shift to sub-optimal cultureconditions; (2) altering the length of time of host cell culture at asub-optimal temperature; and, (3) coordinating the time of infecting thecell culture with virus with a return to an optimal cell culturecondition at or near the known physiological optimum for the respectivehost cell/virus system, this temperature shift occurring at a reasonablepoint in time surrounding the time of virus seeding, namely prior tovirus infection of the cell culture, simultaneous to virus infection orat a point in time subsequent to virus infection of the cell culture.

One embodiment of the present invention relates to a cell culturetemperature shift strategy to enhance virus production; wherein thetemperature shift comprises a lowering of the cell culture temperatureto a level sub-optimal for cell growth for a period of time prior toinfecting the cells with the respective virus, such as a recombinantadenovirus vector. At or near the time of virus infection, thetemperature is again shifted to the physiological cell culturetemperature, or a temperature which approximates the physiologicalculture temperature; the combination of cell exposure at the lowtemperature and the shift upwards reflecting a physiologically optimumpractice for virus production.

Another embodiment of the present invention relates to a cell culturetemperature shift strategy to enhance virus production; wherein thetemperature shift comprises a lowering of the cell culture temperatureto a level sub-optimal for cell growth from the time of inoculating thecell culture with host cells from cryopreserved cells and continuinggrowth of the cell culture at a sub-optimal temperature for one or morethan one passages until a temperature shift to an optimal temperature,which should occur at a reasonable point in time surrounding the time ofvirus seeding, namely prior to virus infection of the cell culture,simultaneous to virus infection or at a point in time subsequent tovirus infection of the cell culture.

To this end, the present invention relates to a method of producingvirus which comprises inoculating and culturing host cells in anappropriate medium at a temperature below a physiological optimum forhost cell growth, infecting the host cells with a virus, resulting invirus-infected host cells, culturing the virus-infected host cells at ornear a physiologically optimum temperature for producing virus,harvesting and lysing host cells, and then purifying virus away from theharvested, lysed host cells, resulting in a purified virus product.

A specific embodiment of the present invention relates to a method ofproducing virus which comprises inoculating and culturing host cells inan appropriate medium at a temperature at or near a physiologicaloptimum for host cell growth, shifting the temperature of the host cellculture to a temperature below a physiological optimum for host cellgrowth, infecting the host cells with a virus, resulting invirus-infected host cells, culturing the virus-infected host cells at ornear a physiologically optimum temperature for producing virus,harvesting and lysing host cells and purifying virus away from theharvested, lysed host cells, which results in a purified virus product.

The time frame for a shift to a suboptimal temperature is preferablyfrom about 4 hours to the entire pre-infection culture period (includingfrom the time of inoculating the culture media with the host cells viainoculation of a cryopreserved ampule of cells) prior to the infectionstep; including but not limited to an initial culture inoculation at asuboptimal temperature, one to several cell passages at a suboptimaltemperature, followed by a temperature shift up to a physiologicallyoptimum temperature for virus infection and production.

The methodology disclosed herein and as summarized in part in theprevious paragraphs of this section are preferably applied to anyserotype of a 1^(st) or 2^(nd) generation adenovirus. Preferable pre-and post-infection cell culture temperatures include but are not limitedto a range of from about 31° C. to 35° C. for suboptimal cell growth sand from about 35° C. to 38° C. as a physiological optimum range for anyculture period before (pre-infection) or after (post-infection)infection of the host cell culture with a virus seed stock.

It is an object of the present invention to provide for a simple yeteffective methodology for enhancing virus production in an establishedhost cell/virus production culture system by incorporating a temperaturescheme as disclosed herein for cell growth and virus infection.

It is a further object to provide for such improved virus productionmethodology by incorporating a temperature shift strategy during virusproduction which calls for a shift to a sub-optimal temperature duringcell culture for a period of time prior to seeding the culture withvirus, followed by a second temperature change in the culture systemwhich results in a temperature shift back to or near what would be aphysiological optimum for the respective host cell/virus productionculture system.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic design of multiple passages of PER.C6™ cellsand adenovirus infection at temperatures in roller bottles underserum-free conditions.

FIG. 2A and FIG. 2B show viable cell concentrations of adenovirusinfected PER.C6™ cells cultivated at temperatures of 31, 33, 35, 37, and39° C.: A. Group I with cells grown at 37° C. prior to virus infection;B. Group II with cells grown at 33° C. for 8 days prior to virusinfection.

FIGS. 3A and 3B show viabilities of adenovirus infected PER.C6™ cellscultivated in at temperatures of 31, 33, 35, 37, and 39° C.: A. Group Iwith cells grown at 37° C. prior to virus infection; B. Group II withcells grown at 33° C. for 8 days prior to virus infection.

FIGS. 4A, 4B and 4C shows adenovirus replication kinetics and effects ofculture temperature on virus productivity in PER.C6™ cultures attemperatures of 31, 33, 35, 37, and 39° C.: A. intracellular virusproductivity in Group I with cells grown at 37° C. prior to virusinfection; B. intracellular virus productivity in Group II with cellsgrown at 33° C. for 8 days prior to virus infection; C. virusproductivity ratio of Group II to Group I.

FIG. 5 shows the experimental design with similarities to that in FIG.1, namely to measure the effect of the length of time at a “sub-optimal”temperature has on Ad5gag virus production.

FIG. 6 shows virus production under different temperature schemes fromthe study described in FIG. 5.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of maximizing the productionof a virus which is relatively thermo-stable under culture conditions,typically any non-enveloped virus, such as adenoviruses, parvoviruses,reoviruses, and/or picornaviruses. It is an accepted practice that cellgrowth in culture is typically conducted at the physiologicaltemperature of 37° C. and virus propagation is conducted either at thesame temperature as cell growth or shifted downward to a lowertemperature. The basis for such a production strategy has been thatculture at the physiological temperature allows optimal cell growth butthe optimal temperature for the production of many viruses is usuallylower due to improved productivity and stability. The present inventionis based on a counter intuitive approach involving cell culture/virusproduction temperature ranges which result in a substantial enhancementof thermal stable virus production. More specifically, the presentinvention relates to a cell culture/virus production temperature shiftstrategy whereby culture of host cells are shifted to a sub-optimalculture temperature for a period of time prior to virus infection orcells are grown at a sub-optimal level for the entire cell expansionprocess including one or more than one passages of cell growth fromcryopreserved cells, followed by a shift back to a more optimaltemperature for virus production. Production of a recombinant adenovirusserotype 5 encoding a HIV gag transgene (Ad5gag) is exemplified herein.It is shown herein that a 2-3 fold enhancement in recombinant Ad5gagproduction occurs when the temperature for host cell growth is shifteddown to a sub-optimal level for a period of time prior to the virusinfection. The temperature is shifted back up to optimal levels postvirus infection.

Therefore, the present invention relates to a method of maximizingproduction of a thermo-stable virus based on a cell culture temperatureshift strategy which results in a substantial enhancement of thermalstable virus production. The manipulation of temperature within the cellculture/virus production process disclosed herein relies upon atemperature shift strategy whereby (1) the culture of host cells areshifted to a sub-optimal temperature for a period of time prior to virusinfection or, (2) the host cell culture is inoculated and grown at arespective sub-optimal temperature, followed by a shift back to a moreoptimal growth temperature at or near the time of virus infection of therespective host cells. Adaptation of such a temperature shift strategypresents a simple yet effective method to substantially increaserecoverable virus within a respective host cell/virus production schemewithout the need to further manipulate other culture and/or mediaconditions within an established host cell/virus production scheme.While specific cell culture and virus production conditions aredisclosed within the Example sections herein, it will be within thepurview of the artisan of ordinary skill to utilize this temperatureshift strategy to optimize virus production for other thermo-stableviruses, regardless of the respective host cell/virus combination. Theartisan may, with the present teachings in hand, adapt and optimize atemperature shift strategy which results in the highest possibleincrease in virus production. It is also within the scope of the presentinvention to alter or manipulate culture conditions, media componentsand other such steps or methods which are known to the artisan which maybe used in combination with a temperature shift strategy. Suchparameters include but are not limited to altering the range of theshift to sub-optimal culture conditions (e.g., a cell culture shift from37° C. to 33° C. and back to 37° C. vs. 37° C. to 31° C. and back to 37°C.), the length of time of host cell culture at a sub-optimaltemperature (e.g., from the time of inoculation, 1 day, 4 days, 20 daysetc.), as well as a coordination of virus infection with a return to anoptimal cell culture conditions (e.g., post-infection, at the time ofinfection, or at a specific time prior to the virus infection).Regardless of the specific parameters adopted, incorporation of atemperature shift strategy will effectively allow for a substantialincrease in virus production versus the utilization of those sameparameters which omit a temperature shift cell culture strategy. Inother words, the temperature shift methodology as disclosed herein willbe especially useful in increasing virus production over and above theproduction levels which exist for a respective host cell/virus system.

In view of the discussion, supra, and the Example sections, infra, thepresent invention relates to a cell culture temperature shift strategyto enhance virus production; wherein the temperature shift comprises alowering of the cell culture temperature to a sub-optimal level for aperiod of time prior to contacting the cells with the respective virus,such as a recombinant adenovirus vector. At or near the time of virusinfection, the temperature is again shifted to the physiological cellculture temperature, or a temperature which approximates thephysiological culture temperature. As exemplified herein, production ofa recombinant adenovirus vector is optimized using a temperature shiftstrategy. Host cells are grown at the optimal growth temperature rangefrom about 35-38° C., more preferably at about 36-38° C., and especiallyat 37° C. at early passages to allow rapid expansion of cell numbers forlarge scale production, which reduces the duration of the batch cycle.The cell growth temperature is then shifted down to a sub-optimaltemperature within a range from about 31° C. to about 35° C. (e.g., 33°C.) and maintained for up to several days prior to the virus infection.After the virus infection, the temperature is shifted up to into the35-38° C. range to maximize the virus productivity. This temperatureshift strategy resulted in a significant enhancement (2-3 fold in rollerbottles and ca. 2-fold in controlled 2 L stirred tank bioreactors) involumetric and cell-specific virus productivity as compared to thetraditional approaches of maintaining the same temperature or shiftingthe temperature down post the virus infection. While the presentinvention is exemplified for a recombinant adenovirus 5 serotype, thetemperature shift strategy disclosed herein is applicable to otheradenovirus serotypes, as well as other thermostable viruses, such aspicomaviruses. The invention is especially related to the increasedproduction of all adenovirus serotypes using E1-transformed mammaliancell lines (293, PER.C6, etc.) in all types of culture vessels(T-flasks, roller bottles, Nunc Cell Factories, Cell Cubes, Wavebioreactor, spinner flask, shaker flask, stirred tank bioreactors, etc.)where temperature control is implemented. Therefore, as noted above, thepresent invention relies in part on a temperature shift strategy forcell growth which comprises reducing the culture temperature to asub-optimal level for a period of time prior to virus seeding, followedby a return to or near the physiological temperature for production ofthat particular virus prior to, simultaneous to, or after virusinfection of host cells. Various parameters which may be furthermanipulated in relation to the temperature shift methodology disclosedherein and which fall within the scope of the present invention includebut are not necessarily limited to (1) altering the range of the shiftto sub-optimal culture conditions; (2) altering the length of time ofhost cell culture at a sub-optimal temperature; and (3) coordinating thetime of infecting the cell culture with virus with a return to anoptimal cell culture condition at or near the known physiologicaloptimum for the respective host cell/virus system, this temperatureshift occurring at a reasonable point in time surrounding the time ofvirus seeding, namely prior to virus infection of the cell culture,simultaneous to virus infection or at a point in time subsequent tovirus infection of the cell culture.

In an embodiment of the present invention, the cell growth temperatureis first shifted from an optimal physiological temperature (e.g., fromabout 35° C. to about 38° C.) to a temperature at or below 35° C. for aperiod of time and then returned to culture at a physiologically optimaltemperature at or near the time of virus infection of the cell culture.This regime results in an effective increase in virus production uponinfection and return to culture conditions at optimal levels. Any“suboptimal” temperature at or below 35° C. is contemplated (with rangesfrom 31° C. to 35° C., preferably 31° C. to 34° C., and most preferablyfrom about 31° C. to about 33° C., with a higher range of 33° C. to 35°C. still being useful to the artisan) to practice the invention, as longas the “suboptimal” cell growth temperature supports reasonable cellgrowth and the optimal temperature for cell culture growth is from about35° C. to about 38° C. (again, with about 36-38° C. and then 36.5 to 37°C. representing especially presferred ranges) and as long as there is anincrease of temperature from the cell growth to virus infection. Asnoted above, while a lower sub-optimal temperature may easily be testedby the artisan, a preferred sub-optimal temperature range is from 31° C.to 35° C., with a more preferred range from 31° C. to 34° C., withsub-ranges of 31° C. to 33° C. and/or 33° C. to 35° C. being useful tothe artisan. A preferable temperature shift strategy is one whicheffectively minimizes duration of cell expansion from a vial to largeproductions scale: cells are expanded at the physiological temperatureof 37° C. and shifted to the sub-optimal temperature for a a specifictime, usually for at least 24 hours prior to the virus infection, with areturn to the temperature of cell growth (such as 37° C.) or slightlylower, depending upon the respective host cell and/or virus).

As noted in the previous paragraph, an embodiment of the presentinvention relates to the period of time which the culture is subjectedto sub-optimal growth conditions. The time period can range anywherefrom several hours, to several passages (multiple days), to entire cellexpansion period inoculating the initial culture from a frozen vialcontaining a stock of host cells. Included in the scope of the presentinvention is a scenario, and all in between, whereby the culture isinitially inoculated at the sub-optimal temperature and kept at thislower temperature until seeding with the virus stock. It should also beunderstood that minor manipulations from a sub-optimal temperatureregime (e.g., such as a short term increase in temperature followed by adownward shift back to the “sub-optimal” temperature) and the length ofthe sub-optimal growth, and the time of returning the temperature backto the optimal temperature with respect to the time of virus infection(e.g., increase the temperature several hours after the virus infectionor several hours before the infection, etc) is well within the scope ofthe present invention.

The host cell for use in the temperature shift protocol comprising thepresent invention may be any mammalian cell line which supportsreplication of the respective thermo-stable virus, especially any hostcell line known in the art which will support infection and replicationof a 1^(st) or 2^(nd) generation adenovirus vector. A preferred hostcell is a host cell line which supports infection and replication of anE1 and/or and E1/E3 deleted recombinant adenovirus. As disclosed herein,such a replication-incompetent virus (such an Ad5gag, as exemplifiedherein) requires a helper cell line which complements the Ad5 E1deletion. Any such cell line may be used to generate recombinant virus,with preferred, but not limiting, cell lines including 293 cells,PER.C6™ cells, 911 cells from a human embryonic retinal cell line(Fallaux et al. 1996, Human Gene Therapy 7: 215-222); E1-transformedamniocytes (Schiedner et al. 2000, Human Gene Therapy 11:2105-2116); anE1-transformed A549 cell line for a human lung carcinoma (Imler et al.1996, Gene Therapy 3:75-84) and GH329: HeLa (Gao et al. 2000, Human GeneTherapy 11: 213-219). Such a cell line is transformed to supportreplication and packaging of a respective recombinant adenovirus, suchas an E1 or E1/E3 deleted recombinant adenovirus. Additional cell lineswhich may be utilized in the present invention are again cell lineswhich have been adapted to act as host cells for a particularthermo-stable virus. It is preferable that the cell line be a continuouscell line and more preferable that the source of the cultured cellsoriginate from a non-neoplastic tissue. It is also preferable that thesource be mammalian, most likely from a primate origin, and especiallyof human origin. Again, a preferred cell line is a cell line which isuseful for the propagation of an Ad E1 or E1/E3 deleted recombinantvirus; a recombinant virus which compliment E1-deleted adenovirus vectorincluded cell lines transfected with the gene encoding Ad E1 which havebeen selected for this transformed phenotype, such as 293 cells(epithelial cells from human kidney) and PER.C6™ (human embryonicretinoblasts). Other cell types include but are not limited to HeLacells, A549 cells, KB cells, CKT1 cells, NIH/sT3 cells, Vero cells,Chinese Hamster Ovary (CHO) cells, or any eukaryotic cells which supportthe adenovirus life cycle.

It is preferred, but not necessary, that the culture be a suspensionculture; a suspension culture which is maintained in a suitable mediumwhich supports cell growth, virus infection and virus production. Such asuspension culture is well known in the art and, again, may be modifiedin any number of ways known to the artisan without effecting a usefulincorporation of a temperature shift strategy to increase virusproduction. The culture medium can be subjected to various growthconditions which are suitable for virus production, including but notlimited to batch, fed-batch or continuous perfusion operations tointroduce fresh medium into the culture medium. Again, the culturemedium can be any suitable medium for maintaining, the cells andpermitting the propagation of the respective virus. Numerous examples ofmedia suitable for use in the practice of the present invention, and theprinciples to generate modified or new suitable media, are widely knownin the art. For a review, see Chapter 8 (serum-based media) and Chapter9 (serum-free media) from Culture of Animal Cells: A Manual of BasicTechnique; Ed. Freshen, RI, 2000, Wiley-Lisps, pp. 89-104 and 105-120,respectively. In general, either serum-based or serum free media will bemanipulated to enhance growth of the respective cell line in culture,with a potential for inclusion of any of the following: a selection ofsecreted cellular proteins, diffusable nutrients, amino acids, organicand inorganic salts, vitamins, trace metals, sugars, and lipids as wellas perhaps other compounds such as growth promoting substances (e.g.,cytokines). As seen with the Trade Index at pp. 483-515 of Culture ofAnimal Cells: A Manual of Basic Technique, (id.), the potentialsuppliers of both information and cell culture media are virtuallyendless. A preferable medium used in the context of the presentinvention is a defined medium, such as the medium exemplified herein asEx-Cell 525 medium (from JRH Biosciences, [http//www.jhrbio.com]) and293 SFM II medium (from Invitrogen). It is also desirable that suchmedia are supplemented with glutamine, as disclosed herein.

The virus types which are amenable to the temperature shift strategy ofthe present invention are preferably from two virus families that arenon-enveloped DNA virus that infect human cells. These two virus typesare the Adenoviridae family (including all known adenovirus serotypes,and recombinant virus generated from such an adenovirus serotype) andmembers of the Picomavirus family (e.g., poliovirus, rhinovirus,hepatitis A virus, Foot and Mouth Disease Virus). An adenovirus 5serotype, a member of the Adenoviridae family, is exemplified herein.The term “virus” as used herein is meant to cover any virus which isamenable to completing its replication cycle in the mammalian cell lineof choice. Therefore, this term is certainly meant to include wild typevirus, any genetically modified virus such as an attenuated virus, ormore likely a recombinant virus vector which may be a developmentcandidate for a potential gene therapy and/or DNA vaccinationapplication. Such programs have created a need for large scalemanufacture and purification of clinical-grade virus. A preferredrecombinant virus which is amenable to the improved cell culture/virusproduction parameters disclosed herein are a family of viruses known asthe adenoviruses. The adenoviruses are grouped within the familyAdenoviridae, which are split into the genus Aviadenovirus (birds) andMastadenovirus (human, simian, bovine, equine, porcine, ovine, canineand opossum). Adenovirus are well known in the art and are subject tomany reviews, such as can be found in Fundamental Biology, 3^(rd) Ed.,Fields, B. N., Knipe, D. M., and Howley, P. M., Ed., at Chapter 30, pp.979-1016 (1996), which is hereby incorporated by reference. Of specificinterest in gene vaccination and/or gene therapy applications is the useof a first generation replication incompetent adenovirus, crippled by E1and/or E3 gene deletions, based on any of a number of adenovirusserotypes, such as serotype 5 of adenovirus. An additional type ofvector is referred to as a 2^(nd) generation adenovirus vector, andcommonly includes a class of adenovirus vectors including “gutless”adenovirus vectors. Gutless adenoviral vectors are adenoviral vectorsgenerally devoid of viral protein-coding sequences, frequently withviral proteins supplemented in trans by a helper virus (often anE1-deleted adenovirus) grown with the HD adenovector in a packaging cellline (e.g., PER.C6™). Absent viral proteins these viral vectors can, inthe alternative, be supplemented in trans by a cell line capable ofexpressing the structural and functional adenoviral proteins necessaryfor successful replication, packaging and rescue. The only cis elementsgenerally present on the BD vector are the packaging signal and theinverted terminal repeats (ITRs). Preferably, inclusive of transgene andany exogenous non-transcribed nucleic acid incorporated therein (stufferDNA), the Ad virion is roughly at least 75% of the wild-type genomelength. The Ad virion has been reported to essentially exhibit a lowerpackaging limit of approximately 75% of the wild-type genome length; seeParks & Graham, 1997 J. Virology 71(4):3293-3298. Adenoviral vectorgenomes smaller than 27.7 kb were found to package inefficiently andfrequently undergo rearrangement. Adenovirus has a broad cell tropismincluding professional antigen presenting cells such as macrophages anddendritic cells, can infect (if not replicate in) cells from most animalspecies, and can be produced in large quantities in appropriate humancell lines designed to provide the E1 gene product in trans.

In a series of experiments which effectively exemplify the presentinvention, but in no way suggest a limitation to the scope thereof,PER.C6™ cells grown at 33 and 37° C. were infected with a firstgeneration adenovirus vector expressing HIV-1 gag at temperatures of 31,33, 35, 37, and 39° C. for virus production. The effects of temperatureon the infected cell metabolism and adenovirus production were studied.It was observed that PER.C6™ cell growth became much more sensitive toculture temperature post adenovirus infection (FIGS. 2A and 2B inExample 1). Even at low temperatures, PER.C6 cells still grew well,albeit at a lower rate and maintained high viability at lowtemperatures. As a result of the virus infection and rapid replicationat high temperatures, cell growth was arrested and cell viabilitydecreased rapidly as a result of the virus infection and rapidreplication (FIGS. 3A and 3B in Example 1). Temperature also affectedthe adenovirus replication kinetics and productivity. The physiologicaltemperature of 37° C. supported the fastest virus replication,regardless of the cell growth temperature prior to infection. Peak virusconcentration occurred earlier at higher temperatures. With cells grownat 37° C. prior to infection, the highest intracellular virusconcentration occurred at 35° C. It occurred at 37° C. with cells grownat 33° C. prior to infection. Cell expansion history prior to virusinfection was shown to play a critical role in infected cell metabolismand virus production. Cells grown at 33° C. prior to infection hadsignificantly higher (60% to 200%) virus productivity at alltemperatures post infection. The results demonstrate that culturetemperature is a highly critical process parameter in adenovirusproduction. The temperature shift improvement has been exemplified inboth small scale roller bottle and 2 L stirred tank or vessel bioreactorstudies. As shown herein, again as an example but in no way alimitation, the cell growth rate at a temperature below 35° C. issignificantly reduced but this suboptimal temperature for cell growthprior to virus infection is optimal for subsequent virus production at ahigher temperature (i.e., an upward temperature shift). It is also shownherein that one to two passages of cell growth at such a suboptirnaltemperature is suited well for adenovirus vector production. Thetemperature shift strategy disclosed herein can be easily implemented atany production scale by controlling temperature at different levelsduring the process. At large scale production, it is perceived that cellgrowth temperature during early cell expansion prior to the cell growthand virus infection in the large scale production can be set at theoptimal physical temperature to achieve the fastest cell expansion forreduction of batch cell duration. However, the inventors have shown thata downward shift in temperature during pre-infection cell culture (e.g.,such as seven to sixteen days prior to the planned virus infection), thetemperature for cell growth can be shifted down to 31-35° C., morepreferably from 31° C. to 34° C., with sub-ranges of 31° C. to 33° C.and/or 33° C. to 35° C. remaining very useful, depending upon therepsective cell culture conditions (this could occur in the seedingvessel for the final production vessel and the final production vessel).Immediately after the virus infection, the temperature is shifted up to35-38° C., preferably from 36-38° C. and most preferably from 36-37° C.to achieve optimal virus production, as exemplified in the followingnon-limiting examples. These non-limiting Examples are presented tobetter illustrate the invention.

EXAMPLE 1 Ad5HIV-1 gag Production in PER.C™ Cells

Materials and Methods:

Cell Line and Maintenance—PER.C6™ cells (Fallaux et al., 1998, HumnanGene Thierapy 9:1909-1917, see also U.S. Pat. No. 5,994,128), a humanembryonic retinoblast cell line licensed from Crucell (Leiden, TheNetherlands), were derived by transfecting human embryonic retinoblastcells with an adenovirus type 5 E1 gene using a phosphoglyceratekinasepromoter. The E1 gene expression confers immortalization on the cellsand allows retention of the E1(+) genotype in the absence of a selectivemarker. The cells were adapted to suspension culture, and routinelymaintained in EX-Cell™ 525 serum-free medium (JRH Biosciences, lenexaKS) supplemented with 4 mM L-glutamine (Mediatech Inc., Hemdon Va.), inroller bottles at 37° C. and 5% CO₂/95% air overlay.

Growth of Cells at Various Temperatures—Two roller bottles of PER.C6™cells maintained at 37° C. were pooled and used to plant ten 850 cm²roller bottles (Corning, Cambridge, Mass.) at 4×10⁵ viable cells per mland 200 ml working volume per roller bottle in Ex-Cell™ 525 serum-freemedium supplemented with 4 mM L-glutamine. The bottles were gassed with5% CO₂/95% air and divided randomly into five groups of two bottles eachand incubated for 3 days with a rotation rate of 4 RPM at nominaltemperatures of 31, 33, 35, 37, and 39° C. At the end of the 3-dayincubation, the two roller bottles from each temperature groups werepooled to plant two new roller bottles and incubated at the respectivetemperatures for a second 3-day passage. The cells from the 33 and 37°C. groups were selected to passage a third time in 5 roller bottles pergroup for two days, followed by virus infection (See FIG. 1). The twogroups of five roller bottles derived from cells grown at 37 and 33° C.prior to the infection are designated as Group I and II, respectively.

Virus Seed Stock—A first generation adenovirus type 5 vector (E1 and E3deleted) expressing the p55 gag transgene from HIV-1 (see WO 01/02607),was amplified in PER.C6™ cells.

Viral Infection at Various Temperatures—The 5 roller bottles from the37° C. and 33° C. groups were infected on day 2 post planting. The spentmedium was removed by centrifugation, followed by resuspending the cellpellets in fresh EX-Cell 525™ medium supplemented with 4 mM glutamine. Afixed quantity of the virus stock was added to infect each rollerbottle, resulting in a multiplicity of infection (MOI) of ˜240 viralparticles (VP) per cell. The 5 infected bottles from each group werethen incubated at nominal temperatures of 31, 33, 35, 37, and 39° C.Samples were taken from each roller bottle at 2, 3 and 4 days post virusinfection, and centrifuged to clarify. Aliquots of supernatant wereremoved and stored at −70° C. for assay of extracellular virus. The cellpellets were resuspended in fresh medium to yield a 10-foldconcentration from the original culture, followed by 3 times freeze andthaw. The resulting lysates were then clarified by centrifugation andstored at −70° C. prior to assay for intracellular virus concentrations.

Temperature Monitoring and Control—Controlled-temperature cultivationtook place in water-jacketed incubators (Form a Scientific, MariettaOhio) and a 37° C. warm room (Environmental Specialties, Raleigh N.C.).Nine days prior to commencement of the experiment, the incubators andwarm room were adjusted to their target temperature set-points.Temperature mappings of the incubators and warm room were carried outduring this period to confirm stability of temperature control. Sixbottle positions were defined on roller apparatus in each of theincubators and the warm room. Temperature probes were inserted throughholes in the roller bottle caps, with the bottles in place and rotatingduring measurement. These measures ensured the accuracy of the culturetemperature for the study.

Analytical Methods—Cell concentrations were measured with ahemocytometer and viability was assessed by trypan blue exclusion. Viralparticle (VP) concentrations were measured by anion exchange HPLC (AEXassay), using a technique adapted from Shabram et al. (1997, Human GeneTherapy 8:453-465). The coefficient of variation for the anion exchangeHPLC assay is typically less than 10%. A quantitative PCR based potencyassay was employed to estimate the virus infectivity. Virus samples wereused to infect 293 monolayer cultures in 96 wells. The viral DNA wasextracted from each well at 24 hours post infection and quantified by aPCR method. Virus infectivity was estimated from a standard virus stocktitered by the traditional TCID₅₀ assay.

Calculation Methods—The time integral of viable cell concentration wascalculated by multiplying the average cell concentration with theculture time between two time points. Specific virus productivity wascalculated by dividing the virus concentration by the viable cellconcentration at infection.

Results:

Effects of Temperature on Virus Productivity—All roller bottles weresampled on day 2, 3, and 4 post virus infection for virus concentrationmeasurements. In previous experience, virus concentration at day 1 postinfection was shown to be below the detection limit of the AEX assay andhence no sample was taken on day 1 post infection in an effort to avoida reduction in the culture volume from the roller bottles. The sampleswere concentrated roughly 10-fold by centrifuging down the cells andresuspending the infected cell pellets in a smaller volume of culturemedium. Virus particle concentrations in the cell pellets were measuredby BPLC assay after a 3× freeze/thaw process for virus release. Thevirus particle concentrations in the cell pellets were normalized on aper cell basis as shown in FIG. 4 (FIG. 4A-Group I; FIG. 4B-Group II).In Group I, the highest virus concentration in the cell pellets occurredat 37° C. on day 2 post infection, suggesting that the virus replicationrate was the highest at this temperature. Deviation to either side ofthis optimal temperature resulted in slower virus replication. However,intracellular virus concentration measured from the cell pellets seemedto have peaked earlier at higher temperatures. On day 3 post infection,virus concentration in the cell pellets decreased at both 37 and 39° C.The reduced concentration was presumably a result of release ofintracellular virions into the culture medium as cell viabilitydecreased rapidly and cell lysis occurred. Although the virus replicatedslightly slower at 35° C., virus concentration continued to rise on day3 post infection, exceeding the peak concentration reached at 37° C. aday earlier. The highest intracellular virus concentration on day 4 wasshifted further down to 33° C. At 31° C., the intracellular virusconcentration was only a fraction of those at higher temperatures,although it continued to rise on day 4. However, it was unlikely toreach a significantly higher level due to the poor replication kinetics.

In Group II, the maximum intracellular virus concentration occurred at37° C. on day 2, at 35° C. on day 3, and at 33° C. on day 4, which isexactly the same as Group I. Peak intracellular concentration occurredon day 2 at 37 to 39° C., on day 3 at 33 to 35° C., and on day 4 at 31°C., which is also the same as Group I. However, there are majordifferences between the two groups. First, the highest intracellularvirus concentration occurred at 37° C. on day 2 in Group II versus 35°C. on day 3 in Group I. Second, virus productivity was significantlyhigher in Group II across all temperatures. A head-to-head comparison isshown in FIG. 6C where the virus titer ratios of Group II to Group I onthe same day are plotted for all five different temperatures. On day 2post infection, virus titers in Group II were 60% to 200% higher than inGroup I. The differences were significantly larger at high temperatures.Differences between the two groups became smaller on day 3 and 4 butremained significant.

The virus concentrations in the supernatants were usually below thedetection limit of the HPLC assay. Hence, a more sensitive infectivityassay was employed to measure this virus. Supernatant and cell pelletsamples were measured head-to-head in the same assay in order toestimate the relative distribution of intracellular and extracellularviruses. As expected, a significant amount of virus was released intothe culture medium, especially at the late stage of virus replicationwhen the cell viability was significantly reduced. These data confirmedthe assumption that the reduction in the virus concentration measured inthe cell pellets was primarily due to the virus release into the culturemedium. Although the above discussion on the virus productivity wasbased on measurements in cell pellets only, the same general picture wasobtained when the percentages of virus in the supernatants wereaccounted for.

The effects of culture temperature on the adenovirus infection ofPER.C6™ cells were thoroughly investigated. Profound differences ininfected cell growth and virus production were observed. Cells infectedwith the same MOI but incubated at different temperature post infectionresulted in different growth behavior. At high temperatures (35-39° C.),the adenovirus infection resulted in complete cell growth arrest, butsignificant cell growth post infection was observed at lowertemperatures (31-33° C.) (FIG. 2A for Group I and FIG. 2B for Group II).Cell viability post infection also showed a strong dependency on theculture temperature (FIG. 3A for Group I and FIG. 3B for Group II). Itdecreased rapidly over the course of virus replication at highertemperatures but maintained at reasonably high levels at lowertemperatures. The MOI used for infection should be high enough for asynchronized infection. The limited cytopathic effect at lowertemperatures indicates slow and impaired virus replication, which isconsistent with low virus concentrations measured over four days postinfection.

Temperature also affected adenovirus replication rate dramatically. Thevirus was found to replicate faster at high temperatures, with theoptimal temperature at 37° C. As a result, virus concentration in cellpellet peaked earlier at 37-39° C. than at 31-35° C. However, theoptimal kinetic temperature did not coincide with the maximum virusproductivity. Cells grown at 37° C. prior to the infection produced thehighest virus concentration at 35° C. while cells grown at 33° C. priorto infection produced the highest at 37° C.

A strong correlation between virus release into culture medium and cellviability was observed. This correlation provides a simple and rapidestimate of virus percentage in the medium, which could be employed todevelop harvest strategies, especially in cases where only intracellularvirus can be harvested. Cell growth history was found to havesignificant impact on cell growth, metabolism, and virus productivity.In addition, there were significant differences in the adenovirusreplication kinetics and productivity. Cells grown at 33° C. prior toinfection had 60% to 200% higher productivities at all temperatures.

EXAMPLE 2 Effect of Passage Time at Sub-Optimal Temperature on VirusProduction

Materials and Methods are essentially as described in Example 1.Briefly, FIG. 5 summarizes the experimental design. Two bioreactors wereinoculated with PER.C6® cells in 293 SFM II (Invitrogen, Grand Island,N.Y.) serum-free medium supplemented with 6 mM L-glutamine (BiowhittakerInc., Walkersville, Md.) at 33.0 and 36.5° C. Cells were grown to˜2.5×10⁶ cells/ml and diluted in new bioreactors at the appropriatetemperature. The temperature control scheme for each vessel is depictedin FIG. 5, using 33.0° C. “temperature shifts” ranging from 2 passagesto 4 hours. The cells were infected with a replication-defectiveadenovirus encoding a HIV-1 gag transgene using a multiplicity ofinfection of 70 viral particles per viable cell. The temperature of allreactors was changed to 36.5° C. immediately after infection. Viralconcentration at 48 hours post infection (hpi) from supernatant andTriton-X100 lysed whole broth samples (TL) containing both intracellularand extracellular virus was determined from HPLC assay daily. The virusbulk was then harvested by addition of a cell lysis buffer to releasethe remaining intracellular virus into the supernatant or by releasingintracellular virus using mechanical shear. The resulted whole brothvirus bulk was then further purified through multiple steps for theremoval of cellular debris, host cell proteins and DNA, unpacked viralproteins and DNA, and other impurities. This example reiterates theresults presented in Example 1, namely that studies in both rollerbottles and 2 L bioreactors indicate that controlling the temperature at33.0° C. during cell growth (for two passages) and at 37.0° C. duringinfection enhanced virus production. Cell growth at 33.0° C. is slower(doubling time ˜50 hr) than at 36.5° C. (doubling time ˜30 hr). Thisresults in an increase in total batch time from ˜12 days in bioreactorsto ˜17 days, which lowers the time-specific virus production of afactory. It will be incumbent upon the skilled artisan to optimize therespective system such that optimal virus production is generated from a“sub-optimal” temperature passage while maintaining the enhanced virusproduction seen in previous experiments.

FIG. 6 shows virus production under different temperature controlschemes, namely differing time periods at a sub-optimal temperatureprior to virus seeding and raising the culture temperature back to aphysiological optimum. These data show that a temperature shift of a fewhours does not provide optimal enhancement of the virus productivity.The length of the “sub-optimal cell growth” at a reduced temperature canbe further optimized to minimize the length of the production process.The data are consistent with the results obtained in roller bottles asdescribed in Example 1. A 2-3 fold enhancement in virus productivity isobtained with the temperature shift strategy for sub-optimal incubationtimes ranging from 7 to 16 days as compared to the 36.5° C. control. Itwill be recognized that these data may be reflective of peculiarities ofthe particular experiment, and that a skilled artisan might optimizeculture conditions aside from temperature so as to shorten thesub-optimal incubation time needed for optimal virus growth from therange shown here and to adjusting the time for the temperature shift-uprelative to the virus infection time.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. A method of producing a virus, comprising: a) inoculating andculturing host cells in an appropriate medium at a temperature below aphysiological optimum for host cell growth; b) infecting the host cellswith a virus, resulting in virus-infected host cells; c) culturing thevirus-infected host cells at or near a physiologically optimumtemperature for producing virus; d) harvesting virus and/or cellscontaining virus from the culture; and, e) purifying virus away fromhost cell and culture contaminants, resulting in a purified virusproduct.
 2. A method of producing a virus, comprising: a) inoculatingand culturing host cells in an appropriate medium at a temperature at ornear a physiological optimum for host cell growth; b) shifting thetemperature of the host cell culture of step a) to a temperature below aphysiological optimum for host cell growth; c) infecting the host cellsof step b) with a virus, resulting in virus-infected host cells; d)culturing the virus-infected host cells at or near a physiologicallyoptimum temperature for producing virus; e) harvesting virus and/orcells containing virus from the culture; and, f) purifying virus awayfrom host cell and culture contaminants, resulting in a purified virusproduct.
 3. A method of according to claim 2 wherein the culturetemperature is lowered to a sub-optimal level for at least about 24hours prior to infecting the host cells with the virus.
 4. A methodaccording to claim 2 wherein the culture temperature is lowered to asub-optimal level for up to the entire cell passages prior to infectingthe host cells with the virus.
 5. A method of producing adenovirus,comprising: a) culturing host cells at a temperature below aphysiological optimum for promoting host cell growth; b) infecting thehost cells with an adenovirus, resulting in adenovirus-infected hostcells; c) culturing the adenovirus-infected host cells at or near aphysiologically optimum temperature for producing adenovirus; d)harvesting virus and/or cells containing virus from the culture; and, e)purifying virus away from host cell and culture contaminants, resultingin a purified virus product.
 6. A method of producing adenovirus,comprising: a) inoculating and culturing host cells in an appropriatemedium at a temperature at or near a physiological optimum for host cellgrowth; b) shifting the temperature of the host cell culture of step a)to a temperature below a physiological optimum for host cell growth; c)infecting the host cells of step b) with a adenovirus, resulting inadenovirus-infected host cells; d) culturing the adenovirus-infectedhost cells at or near a physiologically optimum temperature forproducing adenovirus; d) harvesting virus and/or cells containing virusfrom the culture; and, e) purifying virus away from host cell andculture contaminants, resulting in a purified virus product.
 7. A methodaccording to claim 6 wherein the culture temperature in step b) islowered to a temperature below a physiological optimum for up to theentire cell passages prior to infecting the host cells with theadenovirus.
 8. A method according to claim 6 wherein the culturetemperature in step b) is lowered to a temperature below a physiologicaloptimum for at least 24 hours prior to infecting the host cells with theadenovirus.
 9. A method according to claim 6 wherein the temperature forcell growth in step b) is from between 31° C. and 34° C.
 10. A methodaccording to claim 7 wherein the temperature for cell growth in step b)is from between 31° C. and 34° C.
 11. A method according to claim 8wherein the temperature for cell growth in step b) is from between 31°C. and 34° C.
 12. A method according to claim 7 wherein the temperaturefor cell growth in step a) is from between 35° C. and 38° C. and thetemperature for cell growth in step b) is from between 31° C. and 34° C.13. A method according to claim 8 wherein the temperature for cellgrowth in step a) is from between 35° C. and 38° C. and the temperaturefor cell growth in step b) is from between 31° C. and 34° C.
 14. Amethod according to claim 7 wherein the temperature for cell growth instep a) is from between 35° C. and 38° C. and the temperature for cellgrowth in step b) is from between 31° C. and 34° C. and the temperaturefor growth of infected host cells of step c) is from about 36° C. and38° C.
 15. A method according to claim 8 wherein the temperature forcell growth in step a) is from between 35° C. and 38° C. and thetemperature for cell growth in step b) is from between 31° C. and 34° C.and the temperature for growth of infected host cells of step c) is fromabout 35° C. and 38° C.